Bradford Analysis Research Articles

Bibliometric analysis of studies on gene expression and RNA-seq in cattle species

The aim of this study is to examine the development of research articles on "gene expression and RNA-seq" in cattle species in the Web of Science (WOS) database between 2010 and 2023 using bibliometric mapping methods. Initially, 500 articles were screened using relevant keywords, and 353 articles suitable for analysis were analyzed using the Bibliometrix R package's shiny web application and some analyses were conducted using the VOSviewer application. The conducted analyses included subheadings such as main data information, annual scientific production, countries, and institution analysis, bibliographic coupling with sources, Bradford analysis, Lotka’s law analyasis, highly cited articles, and most influential authors. According to the results obtained after the analysis, it was determined that the institution conducting the most studies in the relevant field is "Universidade De Sao Paulo", the country with the most publications is the USA, and the most published journal is "BMC Genomics". Keyword analysis revealed that the trending topics in recent years are mastitis, dairy cattle farming, and heat stress. The studies were categorized into different clusters related to the reproductive system, immune system and diseases, meat and dairy cattle production. It is recommended that researchers planning to work in this research area on cattle species should design their research, determine the journal to be published, or establish institutional connections by examining the reported study and planning accordingly.

Novel adhesive based on black soldier fly larvae flour for particleboard production

Protein-based adhesives are receiving considerable attention due to their potential to contribute to the development of environmentally friendly materials. Black soldier fly larvae (BSFL) can convert organic waste into high value-added products, such as proteins and fatty acids. The objective of this study is to formulate adhesives from BSFL flour and evaluate their suitability in the production of particleboards. To expose the functional groups of the proteins for reaction with the wood, denaturation strategies involving alkaline treatments were assessed. The effectiveness of these treatments was evaluated through Bradford solubility, SDS-PAGE, and FTIR analysis. Bradford analysis confirmed that the alkali treatment increased the protein solubility from 10.1 g/l for 0 M to 89.1 g/l for 1 M. FTIR analysis revealed changes in the relative intensity of bands attributable to the Amide I, II, and III regions, which are associated with the α-helix and β-sheet structures. The adhesive was applied in the manufacturing process via spraying, requiring an evaluation of the rheological properties of different formulations. Based on the rheological properties, the viscosities of 0.25 M and 1 M were measured at 0.082 Pa·s and 0.113 Pa·s, respectively, falling within the suggested range for particleboard manufacture. Subsequently, eucalyptus wood panels were manufactured using the BSFL-based adhesives, and their mechanical properties as well as water resistance were evaluated. Static bending and tensile strength perpendicular to the surface testing revealed that boards produced with BSFL adhesive exhibited mechanical values comparable to those of commercial resins. The performance of the manufactured materials was compared with the minimum standards outlined in international regulations.

Preparation of isocyanate-containing hydrogel films as antibacterial enzyme immobilization matrices

Sulfobetaine-based isocyanate-containing hydrogel films were synthesized on glass surfaces and the immobilization of small molecules and proteins onto those films was demonstrated. Synthesis of hydrogel films and their functionalizations were confirmed using FTIR spectroscopy. Obtained polymer films were used as matrices for the immobilization of the antibacterial enzyme lysostaphin (Lys). Successful immobilization of Lys enzyme was demonstrated using FTIR and Bradford analyses. The antibacterial activity of enzyme-immobilized surfaces against different Staphylococcus aureus (S. aureus) bacterial strains was evaluated. Enzyme-conjugated surfaces showed 90% killing efficacy against S. aureus 35 bacteria, and the antibacterial activity of those surfaces was maintained for at least 30 days and multiple exposures to the bacteria. The enzyme-immobilized hydrogel coatings reported here provide a promising antibacterial surface against S. aureus.

Exploring the functionality of an active ZrF-laccase biocatalyst towards tartrazine decolorization

This work describes the design and fabrication of zirconia-fucoidan (ZrF) using the modified sol–gel method as the substrate for laccase immobilization (ZrF-laccase). Based on Bradford analysis, the amount of immobilized laccase and the immobilization efficiency were determined at 130 mglaccase/gsupport and 87%, respectively. The immobilization efficiency was also confirmed by determining physicochemical parameters (such as spectroscopic, thermal stability, electrokinetic, surface area and pore volume) of the prepared zirconia-fucoidan materials with and without laccase immobilization. The decrease in the value of the isoelectric point (from 6.4 to 4.0), the specific surface area (from 366 to 108 m2/g) and the pore volume (from 0.33 to 0.18 mL/g) after the immobilization process indicates effective immobilization of the laccase on the zirconia-fucoidan material. The relative activity of immobilized laccase was determined using the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation reaction. The ZrF-laccase biocatalytic system exhibits higher enzymatic activity than free laccase under a range of pH and temperature conditions (above 40% of initial activity) and can be reused in ten reaction cycles, retaining approximately 80% of its initial activity. The ZrF-laccase biocatalytic system was evaluated for the degradation of tartrazine as a model azo dye. The ZrF-laccase biocatalytic system could achieve 95% tartrazine decolorization in five repetitive cycles. Based on mass spectroscopy analysis, the pathway of tartrazine degradation was also proposed. Proposed methodology as well combination of zirconia, fucoidan and laccase appear to be a promising approach and open new pathways for production of highly effective biocatalyst for various applications focused on removal of emerging pollutants from the environment.

Dispersion of ICT-related subject terms in information and knowledge management publications: A Bradford analysis

The application of information and communication technologies (ICTs) in information and knowledge management (IKM) has attracted the attention of library and information Science (LIS) scholars in the recent past. The multidisciplinary and interdisciplinary nature of ICTs means that the ICT literature is published in several fields of knowledge. Although Bradford’s law of dispersion is commonly used to determine the core journals in a given field or discipline or topic of study, this study attempts to use the principles of the law to determine the core concepts of ICTs within the information and knowledge management literature. Data was obtained from EBSCO Discovery’s Library, Information Science and Technology s (LISTA) and the Library and Information Science Source (LISS) databases using a variety of keywords as search terms. The procedures of conducting a Bradford analysis were followed to determine the core subject terms in ICTs as reflected in the IKM group of articles published between 1998 and 2017. The results indicate that the application of ICTs in IKM largely occurs in the fields medicine, business and commerce, education and training (higher education and training), decision sciences, and industrial management. It was also noted that the core subjects varied from one study period to another; technology-associated subject terms were largely visible; and the dispersion of subject terms fits the Bradford’s dispersion. We however noted that the dispersion of articles according to Bradford’s zones is not accurately representative of the principles of the Law. The study has implications for collection development, searching and retrieval practices, and the application of Bradford’s law beyond the analysis of core journals or publications in not only IKM but also in the field of library and information science.

Immunogenicity of multi-walled carbon nanotubes functionalized with recombinant protective antigen domain 4 toward development of a nanovaccine against anthrax

To enhance the stability and immunogenicity of protective antigen (PA) of Bacillus anthracis, Multi-walled Carbon Nanotubes (MWCNTs) as a nanocarrier, was subjected to develop a new PA domain 4 (PAD4) based nanovaccine against anthrax. PAD4 protein was recombinant expressed in Escherichia coli and purified by Nickel–Nitrilotriacetic Acid (Ni–NTA) column. Purified protein was immobilized/absorbed on MWCNTs by covalently and non-covalently methods. SDS-PAGE, Bradford, TEM, SEM, AFM, FTIR, EDX and Zeta potential analyses confirmed MTWCNTs/rPAD4 conjugates. Immunogenicity examinations were performed in four booster dose for six groups of subcutaneous in mice. Determination of antibody response after second and forth booster indicated high levels of anti-rPAD4 IgG titer in serum compared with the control groups. After second booster anti-rPAD4 IgG level in MWCNTs-rPAD4 groups was significantly higher compared with rPAD4 and rPAD4 with Freund's adjuvant groups. In this booster the highest levels of anti-rPAD4 IgG titer was detected with endpoint of 20480 in covalent rPAD4-MWCNTs conjugate. While after fourth booster there was not significant difference between MWCNTs-rPAD4 and rPAD4 with Freund's adjuvant groups. Finally, the results indicated that MWCNTs with enhancing the rPAD4 stability and having the adjuvants effects can be used as an effective delivery vehicle to PA based vaccine formulation against anthrax.

KARAKTERISTIK KIMIA KONJUGAT ISOLAT PROTEIN KEDELAI-LAKTOSA YANG BERPOTENSI DALAM PENURUNAN ALERGENISITAS

Soy protein isolate (SPI) is a product from soybeans with great functionality thus is widely used as an ingredient in any formulation by food and beverage industries. Glycation is commonly done to improve protein functionality and reduce its allergenicity. This research aims to analyze the influence of incubation time of SPI and lactose on the degree of glycation of SPI-lactose conjugates, free amino acid and lactose bases, and to identify the molecular weight and intensity of the protein band of the SPI-lactose conjugate. SPIs were obtained by pH adjustment. The glycation of SPI and lactose was undertaken at pH 9.5, 95°C for 0, 30, 60 and 90 minutes. The determination of the glycation degree of the SPI-lactose conjugate was carried out using several methods, i.e. TBA-based method, HPLC and Bradford analysis. The profiles of the protein molecular weight was analyzed using SDS-PAGE. The results revealed that longer incubation time induced a greater degree of glycation. SDS-PAGE showed that the soybean sample had 13 protein bands with molecular weights ranging from 11.8 to 170.2 kDa. Glycation of SPI with lactose at 95°C could remove or lower the intensity of protein bands with molecular weight of 66.6; 56.1 and 30.9 kDa, which were presumed to be the major allergens in soybean.

EVALUATION OF RENAL EPITHELIAL CELL PROTEIN UNDER STRESS CONDITION

Objective: Proteins are an important component of cells which are involved in various cellular functions. Different kind of stressing conditions has different responses in the components of the protein synthesis system. Super saturation condition in kidney environment leads to crystallization process. Crystals thus formed injure the surrounding cells and result in reactive oxygen species (ROS) formation. There might be some changes in the protein synthesis when the kidney cells enter in oxidative stress. In the present study, kidney cell lines were exposed to oxidative stress and their proteins were analyzed using Bradford analysis and SDS-PAGE.Methods: Vero cells were obtained from NCCS Pune and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) and maintained in a humidified incubator at 37 °C with 5% CO2. Calcium phosphate (CaP) crystals were prepared by the homogeneous system. After FTIR analysis crystals were used to injure Vero cell line. H2O2 was also used to injure the Vero cells. Intracellular protein was extracted from healthy cells and injured cells (with CaP crystals and H2O2). Ammonium sulfate precipitation method was used for the isolation of extracellular protein from the media of healthy and injured cells. Bradford method was used for the quantitative estimation of protein. Extracted proteins were analyzed by SDS-PAGE.Results: Amount of intracellular and extracellular protein of normal cells was 4.84±0.004µg/ml. Intracellular protein of CaP injured and H2O2 injured cells were 10.59±0.003 µg/ml and 10.78±0.011µg/ml respectively. While extracellular protein of injured cells was nearly 4 µg/ml. Intracellular protein bands ranging from 14.3 to 97.4 kDa was observed in healthy cells. Protein bands of ~40kDa and ~20kDa was absent in H2O2 and CaP injured intracellular protein extract. Two extracellular protein bands of 66kDa and ~60kDa were present in injured cells and healthy cells. Conclusion: When exposed to oxidative stress several proteins are oxidized decreasing the activity of many metabolic pathways. In the present study amount of intracellular protein increases when cells are injured with CaP or H2O2. While extracellular protein remains more or less same in both healthy and injured condition of cells. In SDS-PAGE analysis few bands were missing in the intracellular extract of injured cells. These results indicate that the amount of protein varies when cells are injured with CaP and H2O2.

Detection of Salmonella typhi utilizing bioconjugated fluorescent polymeric nanoparticles

Present work demonstrates effective utilization of functionalized polymeric fluorescent nanoparticles as biosensing probe for the detection of Salmonella typhi bacteria on modified polycarbonate (PC) filters in about 3 h. Antibody modified-PC membranes were incubated with contaminated bacterial water for selective capturing which were detected by synthesized novel bioconjugate probe. Core–shell architecture of polymeric nanoparticles endows them with aqueous stabilization and keto-enolic functionalities making them usable for covalently linking S. typhi antibodies without any crosslinker or activator. Bradford analysis revealed that one nanoparticle has an average of 3.51 × 10−19 g or 21 × 104 bound S. typhi Ab molecules. Analysis of the regions of interest (ROI) in fluorescent micrographs of modified fluoroimmunoassay showed higher detection sensitivity of 5 × 102 cells/mL due to signal amplification unlike conventional naked dye FITC-Ab conjugate. Fluorescence of pyrene dye remained same on immobilization of biomolecules and nanoparticles showed stable fluorescent intensity under prolong exposure to laser owing to protective polymeric layer allowing accurate identification of bacteria. Surface-functionalized PC matrix and fluorescent label NPs permit covalent interactions among biomolecules enhancing signal acquisitions showing higher detection efficiency as compared to conventional microtiter plate-based system. Our novel immunoassay has the potential to be explored as rapid detection method for identifying S. typhi contaminations in water.

Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.

Identification of Core Journals and Analysis of Collaboration Networks Among Researchers in Ibero America

In this paper we survey scientific production published in Ibero American journals in four subject areas: Medicine, Education, Administration & Finance, and Psychology during the period between 1997 and mid-2013, based on data from Redalyc. Two aspects are analysed, firstly we conduct a Bradford analysis of the journals in question and identify those journals condense the majority of relevant articles for a researcher in that particular field. Secondly, by applying tools from network analysis, we map the collaboration networks, per country, of researchers publishing in Ibero America.

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18-T vector to construct recombinant clonal vector pMD-ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA-ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA-ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA-ADPN constructed in this study effectively.

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

Journal based current awareness services for systems librarianship

PurposeThis paper aims to explore the effective ways to utilize current awareness services (CAS) for library and information science (LIS) professionals. It seeks to identify core journals for systems librarianship based on Bradford analysis.Design/methodology/approachThe study explored the methods for CAS based on literature review, followed by Bradford analysis of systems librarianship literature, which was conducted by the quantitative analysis approach.FindingsA core journal list for systems librarianship was built based on Bradford analysis. Three effective ways to utilize CAS by table of contents (TOCs) alerts were identified.Originality/valueTo keep up to date with the latest literature in the field of interest, LIS professionals should take advantage of CAS. The study found three effective ways to utilize CAS by TOCs alerts and identified core journals for systems librarianship. The research can help LIS professionals efficiently navigate the information tsunami to obtain the reliable and relevant information that deserves their time and attention, and stay aware of new developments in their area of interest.

A standard procedure for Bradford analysis and its application to the periodical literature in systems librarianship

PurposeThe purpose of this paper is to develop a standard procedure for Bradford analysis: export citations from research databases to a bibliographic management tool, separate the desired citation fields with bibliographic output styles, and then manipulate the empirical data, formulate graph and analyze linearity with Microsoft Excel.Design/methodology/approachThe methodology mainly employed by the paper is to test the theory of Bradford's law of scattering. Based on research work and experiments, a standard procedure for Bradford analysis is to be developed.FindingsA standard procedure for Bradford analysis is developed. The study also shows that the literature in systems librarianship follows Bradford distribution.Originality/valueMany fields or disciplines of literature have been found to follow Bradford's law, which seems to offer a great potential promise for solving library problems. However, empirical Bradford analysis demands tedious computation, and sophisticated graphical formulation. This paper develops an easy operational procedure for Bradford analysis, which will shed light on problem solving in a similar bibliometric analysis.

The research core of the knowledge management literature

A bibliometric analysis and a content analysis were conducted to explore the nature of the knowledge management literature. For the bibliometric analysis, three levels of Bradford analysis were used to examine the shape of the knowledge management literature based on 21,596 references from 2771 source publications. Each of the three analyses conformed to the typical curve of the Bradford distribution. For the content analysis, the texts of 630 knowledge management articles were analyzed to address the question of what research methodologies are used in the knowledge management literature. It was found that 27.8 percent of knowledge management-related articles in knowledge management journals used no identifiable research method. Of the remaining 455 refereed articles, 60 percent employed mainstream social sciences research methodologies. The remaining 40 percent of the articles using an identifiable methodology were characterized by the use of “provisional methods” that appeared to substitute for more formally defined or scientifically based research methodologies.

Stability of Human Salivary Lactate Dehydrogenase in the Present of Ethylenediaminetetraacetic Acid, Glycerol and Polyethylene Glycol at Various Temperatures: Preliminary Study

This study aims to determine the best storage condition for salivary lactate dehydrogenase due to decreased in native enzyme’s activity after seven days of storage. Saliva samples were collected from five healthy and good oral hygiene patients. The specific enzyme activities were measured after 0 (control), 1 and 2 weeks of storage treated with 2.5 mM ethylenediaminetetraacetic acid, 10% (v/v) glycerol or 15% (w/v) polyethylene glycol at three different temperatures, i.e., room temperature, 4 and -20°C. Enzyme activity (unit mL-1) was based on the rate of Nicotinamide Adenine Dinucleotide oxidations standardized at 30°C. The rate of oxidation directly proportional to enzyme’s activity was measured at 340 nm. The specific activity (unit mg-1) was determined through estimated protein content using Bradford analysis. The data were statistically analyzed with paired t-test based on average percentage of enzyme activities from three independent experiments. After two weeks, saliva sample in the presence of polyethylene glycol showed no significant different (p>0.01) at all three temperatures compared to Lactate dehydrogenase basal activity. Lactate dehydrogenase activity of sample in the presence of ethylenediaminetetraacetic acid remained stable (p>0.01) only after a week at room temperature. On the other hand, glycerol managed to stabilize salivary lactate dehydrogenase activity for two weeks at 4 and -20°C. As conclusion, polyethylene glycol showed as the best additive for salivary lactate dehydrogenase storage whereas, ethylenediaminetetraacetic acid suitable only at room temperature for a week. In addition, glycerol was suitable only in cooler conditions.

Mass spectrometry-based proteomic analyses in contact lens-related dry eye.

To identify potential protein biomarkers associated with dry eye in contact lens wearers. Upon enrollment, current galyfilcon A contact lens wearers completed a previously described questionnaire used to classify dry eye status. Approximately 5 microL of aqueous tears were carefully sampled from the inferior-lateral tear prism of each eye using glass microcapillaries. A variety of proteomic approaches were used to compare samples including quantification by Bradford analyses, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (alone in varying percentages and with MultiPlex analyses for posttranslational modifications), nano-liquid chromatography tandem mass spectometry (nano-LC-MS/MS), and differential gel electrophoresis. Twenty-one subjects were enrolled in the study (age 31.3 +/- 11.6 years). Eleven of the subjects were classified with contact lens-related dry eye, while the remaining 10 were normal contact lens wearers. Across all proteomic approaches, several proteins (including several glycoproteins) were identified as potential biomarkers associated with dry eye disease state. In summary across the approaches used, extracellular proteins identified to be altered included beta-2 microglobulin, proline rich 4, lacritin, and secretoglobin 1D1, which were found to be decreased in the dry eye state. Secretoglobin 2A2, serum albumin, glycoprotein 340, and prolactin-inducible protein were all found to be increased in the dry eye state. Dry eye in contact lens wearers is related to several changes in the tear film protein. While functional studies for these candidate proteins are ongoing, initial insights into the functions of these proteins suggest roles in altered tear secretion, in addition to possible increased susceptibility to infection.

Focal Adhesion Kinase as a RhoA-activable Signaling Scaffold Mediating Akt Activation and Cardiomyocyte Protection

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

HMSH4-hMSH5 Adenosine Nucleotide Processing and Interactions with Homologous Recombination Machinery

We have previously demonstrated that the human heterodimeric meiosis-specific MutS homologs, hMSH4-hMSH5, bind uniquely to a Holliday Junction and its developmental progenitor (Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004) Mol. Cell 15, 437-451). ATP binding by hMSH4-hMSH5 resulted in the formation of a sliding clamp that dissociated from the Holliday Junction crossover region embracing two duplex DNA arms. The loading of multiple hMSH4-hMSH5 sliding clamps was anticipated to stabilize the interaction between parental chromosomes during meiosis double-stranded break repair. Here we have identified the interaction region between the individual subunits of hMSH4-hMSH5 that are likely involved in clamp formation and show that each subunit of the heterodimer binds ATP. We have determined that ADP-->ATP exchange is uniquely provoked by Holliday Junction recognition. Moreover, the hydrolysis of ATP by hMSH4-hMSH5 appears to occur after the complex transits the open ends of model Holliday Junction oligonucleotides. Finally, we have identified several components of the double-stranded break repair machinery that strongly interact with hMSH4-hMSH5. These results further underline the function(s) and interactors of hMSH4-hMSH5 that ensure accurate chromosomal repair and segregation during meiosis.